chapter 13: rna processing and post-transcriptional gene regulation

Chapter 13:

RNA Processing and Post-Transcriptional

Gene Regulation

The discovery of split genes along with some other findings of recent years, shows that the genetic apparatus of the cell is more complex, more variable, and more dynamic than any of us had suspected.

Pierre Chambon, Scientific American (1981) 244:60

13.1 Introduction

• Scientists who study RNA have been faced with more revolutionary and unexpected discoveries in the past several decades than in any other area of molecular biology.

• A recurring theme in processing events that involve RNA cofactors is that the RNA is used to provide specificity by complementary base pairing.

13.2 The discovery of split genes

• 1977: The discovery of split genes, first in adenovirus and then in a number of cellular genes.

• Example: The ovalbumin gene was shown by R loop mapping to be split into eight sets of sequences.

• 1978: Walter Gilbert coined the term intron for the intervening sequences that split genes.

• Until the early 1990s, the view was that introns are “junk” that is excised and degraded.

Now known that introns can be functional

• May contain transcriptional regulatory elements.

• May code for small nucleolar RNAs and microRNAs.

Modern definitions

• Introns are sequences that remain physically separated after excision.

• Exons are sequences that are ligated together after excision.

• RNA splicing is the process by which introns are removed from a primary RNA transcript at precisely defined splice sites.

Intron-encoded small nucleolar RNAs and “inside-out” genes

• snoRNAs mediate base modification of ribosomal RNA (rRNA) and possibly other RNAs.

• By complementary base pairing with the rRNA, the snoRNAs recruit the modification enzymes to their target sites.

• Box C/D snoRNAs guide 2′-O-methylation.

• H/ACA snoRNAs direct pseudouridine formation.

• In some “inside-out” genes, the introns code for function and the exons are degraded.

• In 1994, U22 snoRNA and seven other snoRNAs were discovered within separate introns of the U22 host gene.

13.3 Splicing occurs by a variety of mechanisms

Five major classes of introns are distinguished by their structure and mechanisms of splicing

• Autocatalytic group I introns• Autocatalytic group II introns• Archael introns• tRNA introns• Spliceosomal introns in nuclear pre-mRNA

Group I and group II self-splicing introns

• Large catalytic RNAs distinguished by their structure and mechanisms of splicing.

• Both types of introns have mobile members.

Group I introns require an external G cofactor for splicing

• First characterized in Tetrahymena.

• Widely distributed in the mitochondrial, chloroplast, and nuclear genomes of diverse eukaryotes.

• In animals, group I introns have only been found so far in the mitochondrial genomes of one sea anemone and a coral.

Two-step splicing reaction, involving two transesterification reactions

1.Attack by an external guanine on the 5′ splice site, adding the G to the 5′ end of the intron and releasing the first exon.

2.The first exon attacks the 3′ splice site, ligating the two exons together and releasing the linear intron.

• Although many group I introns can self-splice in vitro, many, if not all, require proteins in vivo to fold into the catalytically active structure.

• Proteins required for splicing are either encoded by the introns themselves or by other genes of the host organism.

Group I intron structure

• The typical secondary structure is approximately 10 base-paired helical elements organized into three stacked domains.

Group II introns require an internal bulged A for splicing

• Less common than group I introns.

• Found in the mitochondrial and chloroplast, genomes of certain protists, fungi, algae, and plants, and in bacterial genomes.

First transesterification reaction • Attack by the 2′-OH of an internal bulged A on

the 5′ splice site.

• Release of the first exon.

• Formation of a lariat structure with a 2′→5′ phosphodiester bond.

Second transesterification reaction

• The first exon attacks the 3′ splice site.

• The two exons are ligated together.

• The lariat intron is released, debranched, and degraded.

• Although many group II introns can self-splice in vitro, many, if not all, require proteins in vivo to fold into the catalytically active structure.

• Proteins required for splicing are either encoded by the introns themselves or by other genes of the host organism.

Group II intron structure

• Conserved tertiary structure.

• Six major domains radiating from a central wheel.

Mobile group I and group II introns

• Spread efficiently into a homologous position in an allele that lacks the intron.

• Movement is mediated by highly-specific homing endonucleases that are typically encoded by the self-splicing intron.

• Group II introns also encode an open reading frame with homology to reverse transcriptase.

• Group I introns typically move by a process termed homing.

• Group II introns typically move by a process termed retrohoming.

Archael introns are spliced by an endoribonuclease

• Archaea carry introns in their tRNA, rRNA, and mRNA that are spliced by an archael-specific mechanism.

• Cut-and-rejoin mechanism that requires ATP, an endoribonuclease, and a ligase.

• Bulge-helix-bulge motif at the exon-intron junction.

Some nuclear tRNA genes contain an intron

• Presence or absence of an intron defines two classes of eukaryotic nuclear tRNA genes.

• In humans, only tRNATyr and tRNALeu contain introns.

Nuclear tRNA splicing involves two main reactions:

1. Cleavage: The intron-containing pre-tRNAs are cleaved by an endoribonuclease at the 5′ and 3′ boundaries of the intron.

2. Joining: The paired tRNA halves are joined by ligase.

• In plants and fungi, the 2′ phosphate and 3′-OH at the splice junction are resolved by phosphotransferase.

13.4 Cotranscriptional processing of nuclear pre-mRNA

Eukaryotic mRNA is covalently processed in three ways prior to export from the nucleus:

1. Transcripts are capped at their 5′ end with a methylated guanosine nucleotide.

2. Introns are removed by splicing.

3. 3′ ends are cleaved and extended with a poly(A) tail.

Cotranscriptional processing of nuclear pre-mRNA

• The C-terminal domain (CTD) of RNA polymerase II functions as a “landing pad” for RNA processing factors.

• Deletion of the RNA polymerase II CTD inhibits capping, splicing, and poly(A) cleavage.

• Experiments have shown that different regions of the CTD serve distinct functions in pre-mRNA processing.

• The C-terminal heptapeptide repeats support capping, splicing, and 3′ processing.

• The amino-terminal repeats only support capping.

Addition of the 5′-7-methylguanosine cap

• The cap protects mRNA from degradation and enhances the efficiency of splicing, nuclear export, and translation.

• A distinguishing chemical feature of the cap is the 5′→5′ linkage of 7-methylguanosine to the initial nucleotide of the mRNA.

• Often abbreviated as m7GpppN to reflect this linkage.

The cap is added in three main steps:

• An RNA triphosphatase removes the terminal phosphate from the pre-mRNA.

• Guanylyltransferase adds the capping GMP from GTP.

• Methyltransferases methylate the N-7 of the capping guanosine and the 2′-O-methyl group of the penultimate nucleotide.

Termination and polyadenylation

• No consensus termination sequence has been identified.

• Most metazoan mRNA 3′ ends are produced by cleavage of the pre-mRNA at the polyadenylation site between conserved AAUAAA and G/U-rich sequence elements.

• These regions are recognized by cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF).

• Cleavage requires two additional complexes, mammalian cleavage factor I and II, CFIm, CFIIm.

• The mRNA is cleaved while it is still being synthesized.

• Cleavage also occurs at a cotranscriptional cleavage site (CoTC) which, at least in the case of the -globin mRNA, is self-cleaving.

• The remaining transcript still associated with RNA polymerase II is attacked by the Xrn2 exonuclease that chases after the polymerase.

• When the exonuclease catches up, transcription is terminated.

• The catalytic CoTC core of the -globin pre-mRNA folds into a defined secondary structure.

• The minimal catalytic core was shown to self-cleave in a time course conducted under protein-free conditions.

• After cleavage and release of the mRNA, the transcript is polyadenylated at the 3′ end.

• Most eukaryotic mRNAs have a chain of A residues about 100 to 250 nt long.

• Histone mRNAs have a conserved stem-loop structure instead of a poly(A) tail.

• The poly(A) tail is added by poly(A) polymerase.

• Enhances mRNA stability and translation efficiency.

• The poly(A) tails are coated with sequence-specific poly(A)-binding proteins.

• In the nucleus, PABPN1 increases the processivity of poly(A) polymerase.

• In the cytoplasm, PABPC functions in the initiation of translation and the regulation of mRNA decay.

Oculopharyngeal muscular dystrophy: trinucleotide repeat expansion in a poly(A)-

binding protein gene

• Muscular dystrophy that begins in the eyes and throat.

• Prevalence is highest in French-Canadians who are descended from French immigrants, a man and wife, who emigrated to Canada in 1634.

• Trinucleotide repeat expansion in exon 1 of the PABPN1 gene.

• Normal protein expanded from 10 alanines in a row to 12-17 alanines.

• Leads to intranuclear protein aggregation.

Splicing

• Basic mechanisms is the same as self-splicing group II introns.

• Except, the 3-D structure required for splicing is generated by the spliceosome.

Components of the spliceosome

• Five uracil-rich small nuclear RNA (snRNA)-protein complexes termed snRNPs.

U1, U2, U4, U5, U6

What happened to U3?

• >200 proteins.

The structure of snRNPs

• snRNAs are associated with Sm or Sm-like common core proteins and particle-specific proteins.

• Sm proteins are named after the patient whose autoimmune serum was first used to detect them.

• Sm proteins form a doughnut-shaped complex.

Spinal muscular atrophy: defects in snRNP biogenesis

• Fatal autosomal recessive diseases.

• Degeneration of motor neurons in the anterior horn of the spinal cord.

• Mutations in the Survival of Motor Neurons 1 (SMN) gene.

• Defects in snRNP biogenesis.

SMN-mediated snRNP assembly in the cytoplasm

• Assembly of snRNPs is a stepwise process that takes place in multiple subcellular compartments.

• SMN mediates snRNP assembly in the cytoplasm.

• SMN forms a pre-import complex with snRNPs, importin-1 and the import adaptor snurportin.

SMN is associated with Cajal bodies and gems in the nucleus

• The snRNPs enter the nucleus and are targeted to Cajal bodies.

• Cajal bodies have numerous roles in the assembly and/or modification of the nuclear transcription and RNA processing machinery.

• Splicing takes place at other locations.

Why do defects in SMN lead to spinal muscular atrophy?

• SMN is normally expressed at particularly high levels in motor neurons.

• Neuronal differentiation and maintenance may have particularly stringent demands for splicing.

Assembly of the splicing machinery

• The splicing machinery is recruited to intron-containing transcripts co-transcriptionally.

• The cap-binding complex is important for this recruitment.

• The “splicing cycle” involves the stepwise assembly of the spliceosome through a series of short-lived intermediate subcomplexes, at least in vitro.

• These subcomplexes can be distinguished by their different mobilities in native gels or density gradients, their snRNP composition, and the stage of processing of the pre-mRNA.

• An affinity purification method was used to show that U1 is the first snRNP to bind to pre-mRNA in a yeast nuclear extract.

• An alternative “penta-snRNP” model has been proposed based on in vivo analysis.

• In this model, intron removal is mediated by pre-existing complexes.

• Another more recent in vivo analysis is more consistent with stepwise recruitment of individual snRNPs, rather than a preformed penta-snRNP.

Splicing pathway

• Processing events involving RNA cofactors use the RNA to provide specificity by complementary base pairing.

Splicing pathway

1. Formation of the E (early) or “commitment” complex

• U1 snRNP binds to the conserved 5′ GU splice site.

• The 65 kD subunit of U2 auxiliary factor (U2AF) binds to the polypyrimidine tract.

• The 35 kDa subunit of U2AF binds to the 3′ AG splice site.

2. Formation of the A complex or “pre-spliceosome”

• U2 snRNP binds to the branch site region.

3. Formation of the B complex

• The A complex is joined by the U4/U6/U5 tri-snRNP.

4. Formation of the C complex or “spliceosome”

• Rearrangement of the B complex.• U1 and U4 snRNPs are lost from the complex.• U6 snRNP base pairs at the 5′ splice site and

with U2 snRNP.• U5 snRNP interacts with sequences in the 5 ′

and 3′ exon.

5. Splicing proceeds by two transesterification reactions

• Lariat-shaped intermediate as in group II intron splicing.

• Ligation of the two exons and release of the intron.

• Spliceosome is disassembled.• The lariat intron is debranched and degraded.

Protein factors that help mediate splicing

• Dynamic remodeling of the spliceosome requires DEXH/D box RNA helicases.

• SC35 and ASF/SF2, members of the highly conserved serine/arginine (SR)-rich family of splicing factors have a dual role in stimulating constitutive and regulated splicing.

• SR proteins have a modular domain structure:

– One or two N-terminal RNA recognition motifs (RRMs) for sequence-specific RNA binding.

– C-terminal “RS domain”: repeated arginine-serine dipeptides that can be phosphorylated at multiple positions.

• In the interphase nucleus, the bulk of SR proteins are located in “speckles.”

A model for the regulation of splicing

• The recruitment of splicing factors is controlled by activating and inhibitory splice regulatory proteins.

• The splice regulatory proteins bind exonic and intronic splicing enhancers and silencers.

The U12-type intron splicing pathway

• A minor class of introns (0.15 to 0.34%).

• Variant but highly conserved 5′ splice sites and branch points .

• Spliced with the help of variant classes of snRNAs, including U11, U12, U4atac, and U6atac.

Is the spliceosome a ribozyme?

• Only a few components of the spliceosome interact directly with the pre-mRNA substrate: protein Prp8, U2 snRNA, and U6 snRNA.

• Prp8, a component of the U5 snRNP, is proposed to function as a cofactor in RNA-mediated catalysis.

• There is strong experimental evidence that U2 and U6 snRNA contribute to the catalysis of pre-mRNA splicing.

Experimental Example

• Formation of “RNA X”, a product similar to the first step of splicing, is mediated by U2 and U6 snRNA in vitro.

Prp8 gene mutations cause retinitis pigmentosum

• Genetic disorder causing degeneration of photoreceptors in the retina.

• Severe autosomal dominant form associated with mutations in the gene encoding Prp8, an essential component of the spliceosome.

• Rod photoreceptor cells may be particularly sensitive to alterations in the splicing machinery because of their rapid turnover.

13.5 Alternative splicing

• A versatile means of regulating gene expression.

• Mechanism for generating protein diversity from a small set of genes.

Alternative splicing

• A typical human or mouse gene contains 8-10 exons which can be joined in different arrangements by alternative splicing.

• Splicing of most exons is constitutive.

• Splicing of some exons is regulated.

• At least 74% of human genes are alternatively spliced.

Effects of alternative splicing on gene expression

• Multiple alternative splicing positions in pre-mRNAs gives rise to a family of related proteins.

• Alternative splicing of 5′ or 3′ untranslated regions affects elements that regulate translation, stability, or mRNA localization.

• Insertion of premature termination codons leads to nonsense-mediated decay.

The DSCAM gene: extreme alternative splicing

• Drosophila gene encoding the Downs syndrome cell adhesion molecule (Dscam), a transmembrane neuronal cell adhesion molecule.

• 96 variable exons out of a total of 115.• 38,016 potential different protein isoforms.• Human DSCAM gene has 30 exons and only

three alternatively spliced transcripts.

Regulation of alternative splicing

• Cis-acting regulatory sequences are bound by trans-acting proteins that regulate splicing.

• Exonic or intronic splicing enhancers.

• Exonic or intronic splicing silencers.

• Histone modifications may affect splicing outcome.

Alternative splicing in mammalian heart development

Alternative splicing of Ca2+/calmodulin-dependent kinase II (CaMKII )

• Neuronal isoform (A) is targeted to the transverse tubules of the sarcolemmal membranes.

• Cardiac isoform (B) is targeted to the nucleus.

• Cardiac isoform (C) is located in the cytoplasm of muscle cells.

• In the developing fetal heart, all three isoforms are expressed.

• Between 1-2 months, only the cardiac isoforms are expressed.

• Inappropriate expression of the neuronal isoform (A) leads to defects in heart development and function.

• The exon from one pre-mRNA joins to an exon from another pre-RNA.

• A rare event.

• Occurs in special situations in organisms as diverse as flatworms, the protist Euglena gracilis, plant organelles, nematodes, and Drosophila.

Trans-splicing

Trans-splicing

Three major types

• Discontinuous group II trans-splicing

• Spliced leader trans-splicing

• tRNA trans-splicing

Discontinuous group II trans-splicing

• First discovered in plant chloroplast genomes.

• Coding sequences are joined from separate transcripts to form a complete opening reading frame.

• Example: Trans-splicing events in NADH dehydrogenase 1 (nad1) pre-mRNA

Spliced leader trans-splicing

• First discovered in African trypanosomes.

• A short sequence is added to the 5′ termini of mRNAs.

• The leader exon functions to:– provide a 5′ cap structure.– resolve polycistronic transcripts into individual

capped RNAs.– enhance mRNA translational efficiency.

tRNA trans-splicing

• In Nanoarchaeum equitans, nine genes encode separate tRNA halves which are then joined by trans-splicing.

13.6 RNA editing

• Post-transcriptional modification of the base sequence of mRNA.

• Widespread mechanism for changing gene-specified codons and thus protein structure and function.

• First discovered in trypanosomes, then found in viruses, plants, slime molds, humans, marsupials, squid, dinoflagellates, etc.

RNA editing in trypanosomes

• Early 1980s: Routine study of mitochondrial DNA of African trypanosomes.

• Major RNA transcripts encoding important electron transport chain enzymes found that contained nucleotides not encoded in the DNA.

• Was “The Central Dogma” in jeopardy?

• Extensive post-transcriptional editing (“pan-editing”) of the cytochrome oxidase III transcript in Trypanosoma brucei.

• Cytochrome-b mRNA edited in procyclic-form parasites in the nutrient-poor environment of the tsetse fly, and unedited in bloodstream forms within mammalian hosts.

Editing events: uridine (U) insertions or deletions

• Correct internal frameshifts.

• Create AUG start codons and UAG/UAA stop codons.

• Create open reading frames.

Guide RNAs

1990: The editing mechanism was discovered

• U-insertions and deletions are mediated by multiple short guide RNAs (gRNAs) that specify the edited sequence.

• Catalyzed by a multiprotein complex called the editosome.

• Pre-edited mRNAs are encoded by the larger maxicircles of the mitochondrial DNA genome (kinetoplast).

• Guide RNAs are encoded by the smaller minicircles.

Mechanism of editing

• Mechanism determined by in vitro editing assays, mainly in mitochondrial extracts from procyclic-form trypanosomes.

• Recent development of an in vitro system from mammalian bloodstream-form trypanosomes recreates complete cycles of both insertion and deletion.

• Multiple gRNAs are required to edit each pre-mRNA transcript.

• gRNAs are precise complementary versions of mature mRNAs in the edited region.

• The gRNA provides specificity by complementary base pairing (including GU base pairs).

• Editing occurs from 3′→5′ along the mRNA.

A series of enzymatic reactions catalyzed by the editosome

1. Anchoring: Anchor duplex forms between the pre-mRNA and the gRNA by complementary base pairing.

2. Cleavage: Cleavage at the first site of mismatch by an endoribonuclease.

3. Uridine insertion or deletion: A U is inserted by a 3′ terminal uridylyl transferase (TUTase) or removed by a U-specific endoribonuclease (ExoUase).

4. Ligation: RNA ends are joined by RNA ligase

5. Repeat of editing cycle: The cycle is repeated until the RNA is fully edited.

RNA editing in mammals

• Two main classes of editing enzymes that deaminate encoded nucleotides.

• Adenosine to inosine (A→I) editing.

• Cytidine to uridine (C→U) editing.

• Regulate a diversity of processes, including aspects of neurotransmission and lipid metabolism.

Adenosine to inosine (A→I) editing

• Catalyzed by double-stranded RNA specific ADAR (adenosine deaminase acting on RNA) family.

• Affects >1600 genes: most editing events are within introns and Alu elements in UTRs.

• Altered editing patterns associated with inflammation, epilepsy, depression, malignant gliomas, and amyotrophic lateral sclerosis.

Structure of ADAR

• Crystal structure revealed an unusual feature.

• The enzyme requires inositol hexakisphosphate as a cofactor.

Amyotrophic lateral sclerosis: a defect in RNA editing?

• “Lou Gehrig’s disease.”

• Selective loss of upper and lower motor neurons starting in mid-life.

• Muscle wasting and progressive paralysis.

• There is no cure.

Familial amyotrotrophic lateral sclerosis (ALS)

• Linked to defects in genes encoding superoxide dismutase 1 (SOD1) and senataxin

Sporadic (nonhereditary) (ALS)• Accounts for most cases of the disease.• Cause remains unknown.

Hypothesis for cause of sporadic ALS

• Defect in RNA editing of AMPA receptors (a subtype of glutamate receptors) due to a reduction in ADAR2 deaminase activity.

• AMPA receptors are composed of four subunits in various combinations.

• Almost all GluR2 mRNA undergoes A→I editing.

• Editing changes glutamate (Q) codon to arginine (R) codon.

• The Ca2+ conductance of AMPA receptors varies depending on whether the edited GluR2 subunit is a component of the receptor.

• AMPA receptors lacking GluR2 or containing unedited GluR2 (GluR2Q) are Ca2+ permeable.

• This leads to excitotoxicity and neuronal death.

Cytidine to uridine (C→U) editing

• Only identified in two gene transcripts in mammals

– Apolipoprotein B editing

– Neurofibromatosis type 1 editing

Apolipoprotein B editing in humans

• ApoB is a plasma protein that plays a key role in the assembly, transport, and metabolism of plasma lipoproteins.

ApoB-100: liver specific

ApoB-48: small intestine

• The two different proteins are products of a single gene generated by RNA editing.

• The apoB gene has 29 exons and 28 introns.

• Within the nucleus, the pre-mRNA undergoes

splicing, polyadenylation, and editing in cells of the small intestine.

• The C→U transition converts a glutamine codon (CAA) to a stop codon (UAA).

• There are 375 CAA triplets in the ApoB gene, of which 100 are in-frame glutamine codons.

• Even so, the editing complex is able to confer almost absolute specificity at the correct CAA.

• Binding of ACF to the RNA transcript positions APOBEC1 over the correct cytidine.

• Development of an in vitro editing assay lead to significant advances in understanding the molecular mechanisms for C→U editing.

13.7 Post-transcriptional gene regulation by RNAi

• The recent discovery of hundreds of genes that encode small regulatory RNA molecules represents another landmark discovery in molecular biology.

• Why were small regulatory RNAs overlooked until just recently?

• RNA interference (RNAi) is a sequence-specific gene-silencing process that occurs at the post-transcriptional level.

• In 2002, the journal Science designated RNAi as the “breakthrough of the year.”

Two major classes of small regulatory RNAs

• MicroRNA (miRNA)

• Small interfering RNA (siRNA)

MicroRNA (miRNA)• Post-transcriptional gene regulation.

• Hetero-silencing: derived from unique genes that specify the silencing of different genes.

• First discovered in the early 1990s in the nematode worm C. elegans.

• Now, hundreds of different human miRNAs have been identified.

Small interfering RNA (siRNA) • Defense of the genome.

• Auto-silencing: guide the silencing of the same genetic locus or a very similar locus from which they originate:

– Viruses.– Transposable elements.– Heterochromatin.– dsRNA inserted into a cell by the

bench scientist.

RNAi is triggered by double-stranded RNA (dsRNA) molecules.

1.Dicer, a specialized RNase III family ribonuclease processes dsRNA into short siRNAs of ~21-26 nt in length with two-nucleotide 3′-overhangs.

2.The siRNAs trigger formation of an RNA-induced silencing complex (RISC).

3. The ATP-dependent unwinding of the siRNA duplex by helicase activity in the RISC loading complex leads to activated RISC.

4. The single-stranded siRNA is used as a guide for target RNA recognition (viral or cellular RNA) and cleavage by Slicer activity.

5. In worms, flies, plants, and fungi, RNA-directed RNA polymerase (RdRP) uses the siRNA antisense strands as primers and makes new dsRNA.

• Many bacteria and archaea also protect themselves from viruses through genetic interference pathways.

• In this case, the small RNAs are encoded by clustered, regularly spaced palindromic repeats, called CRISPR RNAs.

• CRISPR RNAs specify the cleavage of foreign DNA.

The discovery of RNAi

Before RNAi was well-characterized the process was referred to by a number of names:

•Post-transcriptional gene silencing (plants).

•Quelling (fungi)

•RNAi (animals)

Experiments by Andrew Fire, Craig Mello and colleagues in 1998

•Discovered that injecting dsRNA into the body cavity of Caenorhabditis elegans worms silenced only mRNAs containing a complementary sequence.

•Fire and Mello were awarded the Noble Prize in 2006 for their discovery of RNAi.

RNAi machinery

• Gene silencing is carried out by RISC, the RNA-induced silencing complex.

• Experiments have shown that the Argonaute subunit of RISC has “Slicer” activity.

– PAZ (Piwi Argonaute Zwille) domain: RNA binding.

– PIWI domain: nuclease.

The discovery of miRNA in Caenorhabditis elegans

Heterochronic gene hierarchy

• Lin-4 miRNA regulates the timing of larval development in C. elegans.

• Lin-4 miRNA base pairs with the 3′ UTR of lin-14 mRNA and lin-28 mRNA.

• Originally thought to block mRNA translation.

• More recent results suggest that lin-4 miRNA also promotes degradation of lin-14 and lin-28 mRNA.

• When let-7 miRNA was discovered 7 years after lin-4, it became clear that this type of regulatory mechanism was not unique.

• Discovery of RNAi and siRNAs dramatically increased interest in small regulatory RNAs.

Processing of miRNAs

• miRNAs are processed from gene transcripts that form hairpin structures.

• Many human miRNA genes (~56%) are located within the introns of protein-coding pre-mRNAs.

miRNAs biogenesis and function involves six main steps

1. Transcription: The miRNA gene is transcribed, possibly by RNA polymerase II.

2. Cleavage by Drosha: The primary miRNA (pri-miRNA) hairpin is processed by Drosha in the nucleus.

3. Nuclear export: The pre-miRNA exits the nucleus by an exportin 5-mediated pathway.

4. Cleavage by Dicer: The pre-miRNA is cleaved into duplex miRNA by Dicer.

5. RISC formation: The duplex is unwound by a helicase in the RISC loading complex and the mature single-stranded miRNA associates with activated RISC.

6. mRNA inhibition: RISC targets mRNAs for either degradation or translational repression depending on the degree of complementarity.

miRNAs target mRNA for degradation and translational inhibition

• Short complementary regions in 3′-UTR:

– Translational repression

• Extensive complementarity in coding regions or UTR:

– mRNA degradation

• 100s of miRNAs have been cloned or predicted

• Play a role in:

– Apoptosis.– Neuronal asymmetry and brain morphogenesis.– Leaf and flower development.– Development of cancer.– Fortuitous anti-viral defense.– Tissue specific regulation of mRNA levels.

• The function of the majority remains unclear.

Tissue-specific gene expression for genes downregulated by miRNAs

• miRNAs were transfected into human cells and microarray analysis was used to examine changes in mRNA expression profiles.

• Delivery of muscle-specific miR-1 shifted the profile towards that of muscle.

• Delivery of brain-specific miR-124 shifted the profile towards that of brain.

Antiviral defense

• Cellular miRNA was shown to restrict accumulation of primate foamy virus type 1 in human cells.

• Fortuitous recognition of a complementary sequence in the viral RNA by miRNA-32.

13.8 RNA turnover in the nucleus and cytoplasm

• Quality control pathways in the nucleus.

• Some mRNAs are stored in the cytoplasm.

• Most mRNAs are immediately translated and later degraded.

• Nonsense-mediated decay of mRNA.

Nuclear exosomes and quality control

• Short-lived processing intermediates of rRNAs, snoRNAs, and snRNAs are degraded by the nuclear exosome from 3′→5′.

• Specific nuclear decay pathways destroy defective pre-mRNAs or those failing to form export-competent mRNPs.

• The TRAMP complex adds a short poly(A) tail to target molecules before recruitment and activation of the exosome.

• TREX and SR proteins recruit general nuclear export factors, such as TAP/NFX1 to mRNAs.

• Contribute to the ability of the export machinery to discriminate between spliced and unspliced mRNPs.

• Further quality control surveillance and retention of unspliced transcripts occurs at the nuclear pore complex.

Cytoplasmic RNA turnover

• Once in the cytoplasm there are several possible fates for an mRNA:

– Held in a translationally silent state.

– Translated and then degraded.

– Nonsense-mediated mRNA decay.

Storage of translationally silent mRNA

• Mediated by deadenylation at a particular stage of development.

• Re-addition of the poly(A) tail later in development.

General mRNA decay pathways

• Most mRNAs are immediately translated and later degraded by general decay pathways.

• The 5′ cap is removed by decapping enzymes, followed by exonuclease digestion from 5′→3′.

• Alternatively, the 3′ poly(A) tail is removed by deadenylases and the rest of the mRNA is degraded by exonucleases from 3′→5′.

• mRNAs that contain a premature termination codon are rapidly degraded by nonsense-mediated mRNA decay.

• Exon junction complexes (EJCs) are assembled near exon-exon boundaries after mRNA splicing.

Nonsense-mediated mRNA decay

• When the ribosome begins translating an mRNA, the EJCs are normally displaced.

• If a premature termination codon is present, this activates a surveillance complex.

• The surveillance complex interacts with the prematurely terminating ribosome and targets the mRNA for degradation.